Protective Effect of Trillium Tschonoskii Maxim Saponin on … · there is an increasing drug demand for the protection of the liver, modern medicine is still lack of reliable liver-protective

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Protective Effect of Trillium Tschonoskii Maxim Saponin on

CCl4-induced Acute Liver Injury of Rats through Apoptosis Inhibition

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0228.R1

Manuscript Type: Article

Date Submitted by the Author: 27-May-2016

Complete List of Authors: Wu, Hao; Hubei University for Nationalities, College of Science and

Technology Qiu, Yong; Hubei University for Nationalities, College of Medicine Shu, Ziyang; Wuhan Sports University, College of Health Science Zhang, Xu; Wuhan Sports University, College of Health Science Li, Renpeng; Hubei University for Nationalities, College of Science and Technology Liu, Su; Affiliated Hospital of Hubei University for Nationalities Chen, Longquan; Hubei University for Nationalities, College of Medicine Liu, Hong; Hubei University for Nationalities, College of Medicine Chen, Ning; Wuhan Sports University, College of Health Science

Keyword: Trillium tschonoskii Maxim (TTM), liver injury, hepatomegaly, inflammatory factor, apoptosis

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Manuscript type: Original paper

Protective Effect of Trillium Tschonoskii Maxim Saponin on CCl4-induced

Acute Liver Injury of Rats through Apoptosis Inhibition

Hao Wu1, 2, #

, Yong Qiu2, #

, Ziyang Shu3, Xu Zhang

3, Renpeng Li

1, Su Liu

4, Longquan Chen

2,

Hong Liu2, *

, Ning Chen3, *

1College of Science and Technology, Hubei University for Nationalities, Enshi 445000,

China

2College of Medicine, Hubei University for Nationalities, Enshi 445000, China

3Hubei Key Laboratory of Sport Training and Monitoring, College of Health Science, Wuhan

Sports University, Wuhan 430079, China

4Affiliated Hospital of Hubei University for Nationalities, Enshi 445000, China

Running title: TTM against liver injury

#These authors contributed equally to this project.

*Corresponding author:

Ning Chen, PhD, Professor of Biochemistry

College of Health Science, Wuhan Sports University, Wuhan 430079, China

Tel: 86-27-67846140

Fax: 86-27-67846140

E-mail: [email protected]

Hong Liu, Professor of Pharmacology

School of Medicine, Hubei University for Nationalties, Enshi 445000, China

Tel: 86-718-8437479

Fax: 86-718-8437479

E-mail: [email protected]

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Abstract

In order to explore hepatoprotective role and underlying mechanisms of TTM, 36 rats were

randomly divided into control, CCl4-induced liver injury model, and DDB and low, moderate

and high-dose TTM treatment groups. After CCl4-induced model establishment, the rats from

DDB and TTM groups were administrated with DDB at 0.2 g/kg⋅d and TTM at 0.1, 0.5 and

1.0 g/kg⋅d, while the rats from control and model groups were administrated with saline.

Upon 5 days treatments, all rats were sacrificed for determining serum ALT and AST levels

and liver index, examining histopathological changes in liver through HE and TUNEL

staining, and evaluating TNF-α and IL-6 mRNA expression by RT-PCR, and Caspase-3,

Bcl-2 and Bax expression by Western blot. Results indicated that CCl4 could induce acute

liver injury and abnormal liver function in rats with obvious hepatomegaly, increased liver

index, high ALT and AST levels, up-regulated TNF-α and IL-6, and overexpressed Bax and

Caspase-3. However, DDB and TTM could execute protective role in CCl4-induced liver

injury in rats through reducing ALT and AST levels, rescuing hepatomegaly,

down-regulating inflammatory factors and inhibiting hepatocyte apoptosis in a

dose-dependent manner. Therefore, TTM has obvious protective role in CCl4-induced liver

injury of rats through inhibiting hepatocyte apoptosis.

Key words: Trillium tschonoskii Maxim (TTM), liver injury, hepatomegaly, inflammatory

factor, apoptosis.

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Introduction

Liver plays an important role in metabolism and detoxification in human body. It is also

one of the organs sensitive to a series of stimuli such as trauma, viral infection, autoimmune

disease and other exogenous materials including drugs, alcohol and toxins, thus resulting in

its acute or chronic damage (Shi et al. 2014; Wang et al. 2007). Severe acute liver injury

could result in life-threatening clinical problems such as jaundice, serious blood coagulation

disorders and high death rate (Bernal et al. 2010). At the same time, the long-term

accumulated liver injury also can lead to liver fibrosis, cirrhosis and hepatocellular carcinoma

(HCC), so it is very important for the prevention of liver injury (Dong et al. 2016). Although

there is an increasing drug demand for the protection of the liver, modern medicine is still

lack of reliable liver-protective drugs (Lu et al. 2016), and severe acute liver injury is still a

great challenge in the field of clinical medicine (Auzinger and Wendon 2008).

During long application history in China, Chinese herbs or natural products have

obvious treatment efficacy for a series of diseases with little side effects and low cost (Kou

and Chen 2012; Kou et al. 2013; Kou et al. 2015; Zhang et al. 2014). Therefore, more and

more people are exploring a novel strategy for the prevention and treatment of liver diseases

through Chinese herbs (Akhter et al. 2015; Yan et al. 2015).

Trillium tschonoskii Maxim (TTM), a pharmacologically important medicinal plant

containing steroidal saponins and alkaloids from Liliaceae, is widely distributed in central

and western China (Li et al. 2005; Zhang et al. 2013). Its dried rhizome and root have

medicinal roles in promoting blood circulation and stanching bleeding, detoxification,

tranquilizing and allaying excitement, so that it is usually used for the treatment of

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hypertension, neurasthenia, dizziness, headache, traumatic injury and bleeding as well as a

series of liver disorders (Fu 1992). TTM is honored as one of four magical herbs from Tujia

nationality due to its obvious curative effects for a series of diseases. However, as it’s a kind

of medicinal plant from the minority in China, its clinical application is limited in the regions

of Tujia and Miao nationalities without extension all over the China.

According to previous reports, saponins from several herbs are known to induce

apoptosis in cancer cells and are proposed to be promising modulators of drug resistance.

Paris saponin VII extracted from T. tschonoskii can reverse multidrug resistance of

adriamycin-resistant MCF-7/ADR cells via P-glycoprotein inhibition and apoptosis

augmentation (Li et al. 2014), inhibit metastasis by modulating matrix metalloproteinases in

colorectal cancer cells (Fan et al. 2015b) and reduce migration and invasion in human A549

lung cancer cells (Fan et al. 2015a). The saponin TTB2 isolated from T. tschonoskii Maxim

can exert anticancer effects and may be a potential candidate for the development of

anticancer drugs (Huang and Zou 2015). The true incidence of phytotherapy-related

hepatotoxicity and its pathogenic mechanisms are largely unknown (Tarantino et al. 2009). It

is important to increase the awareness of both clinicians and patients about the potential

dangers of herbal remedies. Meanwhile, the role of TTM in liver disorders and underlying

mechanisms are also not fully understood. In order to further confirm its treatment efficacy

and extend its clinical application, we established CCl4-induced acute liver injury model in

rats to explore its protective roles and underlying mechanisms for diseases associated with

acute liver injury.

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Materials and methods

The preparation of TTM saponin

TTM provided by Specimen Center of Hubei University for Nationalities was smashed

after dried in 60 °C constant temperature oven, then sequentially immersed in 75% alcohol

for 2 hours and completed ethanol reflux extraction for three times. The alcohol extract after

filtration was concentrated to 1 g/mL for future use. The saponin in TTM extract was

analyzed by high-performance liquid chromatography to reveal total saponin of 18.54%.

Animal grouping and drug administration

The healthy male rats with body weights of 180-220 g were ordered from Hubei

Research Center of Laboratory Animals (License number: 2015-0018). All rats were

randomly divided into 6 groups including control group, CCl4-induced acute liver injury

model group, biphenyl dimethyl dicarboxylate (DDB) group at the dose of 0.2 g/kg⋅d, and

TTM groups at low, moderate and high dosages (0.1, 0.5 and 1.0 g/kg⋅d), respectively. Six

rats were in each group. All rats were subjected to adaptive feeding with regular feeds for 1

week. The rats were subjected to diet restriction for 12 h prior to model establishment. The

rats from the control group were subjected to subcutaneous injection of peanut oil at the dose

of 5 mL/kg, while the rats from other groups were subjected to the administration of peanut

oil mixed with 25% CCl4 at the dose of 5 mL/kg (equal to 2 g/kg for CCl4) for the

establishment of acute liver injury model. After model establishment, the rats from treatment

groups were administrated with DDB and TTM at the designed dosages through gavage twice

a day with an interval of 12 h for 5 successive days. The diets of the rats were restricted

except water after the last drug administration. Twenty-four hours later, the rats were

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anesthetized with 10% chloraldurate, and blood and liver tissue samples were harvested for

analysis.

Liver index determination

The body weight of each rat was measured prior to being sacrificed. Similarly, the wet

weight of rat liver was measured after sacrificed. The liver index = liver weight/rat body

weight × 100%.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level

measurement

Blood samples of the rats after last drug administration for 12 h were harvested in a

common anticoagulant tube, and then subjected to the centrifugation at the speed of 1000 ×g

for 5 min. The supernatant was collected, and then serum ALT and AST levels were

determined by immunity transmission turbidity method using commercially available assay

kits (Mindray Bio-medical Electronics Co., Ltd, Shenzhen, China) in an automatic

biochemical analyzer (BS-800M, Mindray Bio-medical Electronics Co., Ltd, Shenzhen,

China).

Real-time polymerase chain reaction (RT-PCR) detection

Approximately 0.1 g liver tissue was subjected to complete homogenate. Total RNA was

extracted using Trizon reagent (Invitrogen, USA) following manufacturer’s instructions and

then reversely transcribed into cDNA. The mRNA expression levels of IL-6 and TNF-α were

determined through fluorescence quantitative RT-PCR. The forward and reversed primers for

IL-6 were 5’-GCTCTCCGCAAGAGACTTCCA-3’ and

5’-GGTCTGTTGTGGGTGGTATCC-3’, respectively. The forward and reversed primers for

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TNF-α were 5’-GGTGATTGGTCCCAACA-3’ and 5’-GTCTTTGAGATCCATGCC-3’,

respectively. The forward and reversed primers for β-actin as the internal control were

5’-CACGATGGAGGGGCCGGACTCATC-3’ and

5’-TAAAGACCTCTATGCCAACACAGT-3’, respectively. The relative expression level of

IL-6 or TNF-α was calculated by the ratio of IL-6 or TNF-α to β-actin.

Histology and immunohistochemistry

The small slice of liver tissue from each rat was subjected to the fixing by 4%

paraformaldehyde, regular embedding by paraffin, sectioning, hematoxylin-eosin (HE)

staining and Ki67 staining (Abcam, Cambridge, USA), and the slides were observed for

conventional morphological evaluation under a light microscope (Nikon Eclipse TE2000-U,

NIKON, Japan) and photographed at 100× magnification.

Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining

The sections of liver tissues were analyzed by terminal deoxynucleotidyl transferase

dUTP nick end labeling (TUNEL) assay using the in situ cell death detection kit (Roche

Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.

TUNEL-positive cells were captured under a light microscope (Nikon Eclipse TE2000-U,

NIKON, Japan) and analyzed through Image pro-plus 6.0 software (Media Cybernetics Inc.,

MD, USA).

Apoptosis-related protein expression evaluated by Western blot

Approximately 0.1 g of liver tissue after homogenizing was lysed with RIPA buffer

(Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of PMSF (Beyotime Institute of

Biotechnology, Jiangsu, China) at the speed of 4000 ×g at 4 °C for 20 min. The collected

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supernatant was added with loading buffer and boiled at 95 °C water batch for 5 min. Then,

20 µg total protein was separated on sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE), and transferred to PVDF membrane. The membrane was

blocked with 5% skim milk at room temperature for 3 h. The targeted protein on the

membrane was probed with primary antibody such as Caspase-3, Bax or Bcl-2 (Cell

Signaling Technology, USA, 1:1000) at 4 °C overnight. The membrane was then washed

with TBS-T buffer for three times with 15 min in each time. Sequentially, the membrane was

incubated with secondary antibody, HRP-labeled anti-rabbit IgG (Cell Signaling Technology,

USA, 1:30000) for 2 h, and washed with TBS-T buffer for three times. Then, the

chemiluminescence agent was incubated with the membrane in dark room for 2 min and the

protein probed by primary antibody in the membrane was imaged. The β-actin was used as an

internal reference. The data were analyzed by the software Quantity one 4.6.1 (Bio-Rad

Software Inc., California, USA).

Statistical analysis

All data were expressed as mean ± standard deviation (M ± SD). All statistical analyses

were conducted using SPSS19.0 software (SPSS, Inc., Chicago, USA) and GraphPad Prism

6.0 (GraphPad Software, Inc., San Diego, USA) through ANOVA with post-hoc tests. The

significant difference and very significant difference were considered at P < 0.05 and P <

0.01, respectively.

Results

TTM rescues CCl4-induced acute liver injury

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Serum ALT and AST levels, as the golden biomarkers for liver damage, are

well-accepted indicators to evaluate the severity of liver injury (Shi et al. 2014). In order to

validate the treatment efficacy of TTM on acute liver injury, we established the rat model

with CCl4-induced acute liver injury to explore the treatment efficacy of TTM. Compared

with the control group, the levels of serum ALT and AST in the model group revealed an

obvious increase (P < 0.01 and P < 0.05, respectively), indicating that the establishment of

CCl4-induced acute liver injury model was successful. In contrast, after treatment with DDB

and TTM, the serum ALT levels of the rats treated with DDB and TTM at low, moderate and

high dosages revealed a very significant reduction (P < 0.01); the serum AST levels of the

rats treated with DDB and high-dose TTM exhibited a significant decrease (P < 0.05),

indicating that TTM treatment can decrease serum ALT and AST levels in a dose-dependent

manner (Figures 1A and 1B).

TTM inhibits CCl4-induced hepatomegaly and liver index

In order to explore the effect of TTM on CCl4-induced histopathological change of liver

tissues in rats, we compared fresh liver tissues of the rats from various treatments. As shown

in Figure 2A, the rats from CCl4-induced model group revealed obvious hepatomegaly when

compared with the rats from control group and treatment groups. Subjected to the treatments

with DDB and TTM, the hepatomegaly of the rats exhibited an obvious inhibition when

compared with that of the CCl4-induced model rats, suggesting that both DDB and TTM have

obvious protective roles in CCl4-induced hepatomegaly.

Liver index reflects liver pathological changes and nutritional status in the body (Chen

1993). Based on the results of liver index, its significant difference between model group and

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control group was observed (P < 0.01), suggesting that CCl4 could result in hepatomegaly,

while the treatment with BBD and TTM at various dosages could inhibit CCl4-induced

hepatomegaly in rats (Figure 2B).

In addition, the liver tissues were examined with HE staining. As shown in Figure 2C,

normal liver lobule structure and liver cells in rats from normal control group were observed.

In contrast, in the CCl4-induced model group, diffused edema, necrosis and inflammatory cell

infiltration in liver tissues were observed. However, the treatments with DDB and TTM at

various dosages could rescue the liver damage with the significant reduction of diffused

edema, necrosis and inflammatory cell infiltration in liver tissues when compared with the

model group. Moreover, the protection of TTM on liver tissues revealed an obvious

dose-dependent manner.

TTM reduces inflammatory factors for CCl4-induced acute liver injury

TNF-α and IL-6 are two important pro-inflammatory factors causing cell inflammation

and death (Diehl 2000; Schmich et al. 2011; Tilg 2001). Then, we examined the mRNA

expression of both factors through RT-PCR. Results indicated that acute liver injury induced

by CCl4 could result in an obvious increase of TNF-α and IL-6 (P < 0.05, and P < 0.01,

respectively) (Figures 3A and 3B). On the other hand, after treatment with DDB and TTM at

various dosages, the expression of TNF-α and IL-6 in DDB and TTM groups revealed an

obvious reduction. Therefore, TTM can alleviate inflammatory response of liver cells to

execute the protective function for liver.

TTM inhibits apoptosis of liver cells induced by CCl4

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In order to further understand the protective mechanism of TTM for acute liver injury,

the cell apoptosis in liver tissues from the rats subjected to CCl4 induction and DDB and

TTM treatments at various dosages were examined through TUNEL staining. Compared with

normal control group, a large number of cell apoptosis in liver tissues due to the CCl4

induction in the model group was observed (Figure 4A); in contrast, subjected to the

treatments with DDB and TMM at moderate and high dosages, the apoptotic cell number

revealed a significantly decrease and the reduced apoptosis of liver cells exhibited as a

dose-dependent manner for TTM (Figure 4B). Similarly, compared with the normal control

group, CCl4-induced liver tissues revealed an obviously increased Caspase-3 and Bax and

reduced Bcl-2; on the other hand, the treatments with DDB and TTM at moderate and high

dosages could result in the down-regulation of Caspase-3 and the up-regulation of Bcl-2

when compared with those in CCl4-induced liver injury model group (Figure 4C). Therefore,

the protective role of TTM in acute liver injury should be correlated with the inhibition of

liver cell apoptosis.

Discussion

The mechanisms of the diseases with acute liver injury are very complex. Current reports

are mainly focused on oxidative stress, inflammation and apoptosis (Abouzied et al. 2016;

Chan et al. 2014; Schmich et al. 2011). The CCl4-induced acute liver injury rat model is well

accepted, which can result in substantial liver cell damage and liver dysfunction (Masuda

2006; Weber et al. 2003). Once CCl4 is absorbed by the body, it can activate liver

cytochrome (P450) to generate trichloromethyl free radicals (CC3•) and peroxide

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trichloromethyl free radicals (CCl3O2•), thus attacking phospholipid molecules on cell

membrane of liver, increasing cell membrane permeability, and finally leading to liver cell

swelling, necrosis, and symptoms of acute liver injury (LeSage et al. 1999; Weber et al.

2003).

In the present study, we utilize CCl4-induced rat model with acute liver injury to clarify

the treatment efficacy of TTM and underlying mechanisms. The administration of TTM at

various dosages for model rats with acute liver injury by lavage for five days can reduce the

levels of serum ALT and AST and liver index, and down-regulate the expression of TNF-α

and IL-6. HE staining revealed that TTM treatment could alleviate inflammatory necrosis of

liver tissues, and present an obvious protective effect on liver damage.

It is well known that the liver has strong capacity of regeneration and recovery (Kim and

Kim 2013). Therefore, we suspect TTM protection against acute liver injury may be due to

the promotion from liver cell regeneration. In our study, compared with CCl4 model group,

the expression of Ki67 revealed an obvious increase after the treatment with DDB; in

contrast, no obvious expression of Ki67 was observed in liver tissues from the rats treated

with TTM at various dosages (Figure 5), suggesting that the protective effect of TTM on

acute liver injury may be not correlated with the regeneration of liver cells.

Mitochondrial apoptotic pathway is one of the major pathways of apoptosis. Bcl-2

protein family plays a vital role in mitochondrial apoptotic pathway (Tischner et al. 2010).

Bax, Bcl-2, and Bcl-2 protein family can promote or inhibit the release of cytokines and other

apoptotic factors through changing the mitochondrial outer membrane permeability (Rosse et

al. 1998). Cytochrome C could mediate the activity of Caspase-3, and the activated Caspase-3

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can induce cell apoptosis (Cusack et al. 2013). In the present study, CCl4-induced liver cell

apoptosis could be obviously alleviated in the presence of TTM treatment under the

examination by TUNEL staining; similarly, based on the analysis of Western blot, TTM

could reduce the expression of cleaved Caspase-3 and Bax, and improve the expression of

Bcl-2, thus further verifying the inhibitory role of TTM in apoptosis of liver cells for the

protection of acute liver injury. However, apoptotic cell death can be also associated with

mitochondrial membrane depolarization and cytochrome C release. The exposure of

hepatocytes to hepatototoxic substances may result in increased ROS production and

mitochondrial damage, and is balanced by the presence of antioxidant substances. Therefore,

circulating levels of cytochrome C, gamma-glutamyl transferase, triglycerides and

unconjugated bilirubin, as well as Bcl-2 in overweight/obese patients is highly associated

with non-alcoholic fatty liver diseases (Tarantino et al. 2011a; Tarantino et al. 2011b).

Whether TTM can inhibit the release of cytochrome C, alleviate the production of ROS,

reduce the damage of mitochondria, and be benefit for non-alcoholic fatty liver diseases

needs to be further explored. As the crosstalk between apoptosis and autophagy for

maintaining cellular hemostasis in a series of diseases (Chen and Karantza 2011; Chen and

Karantza-Wadsworth 2009; Fan et al. 2016), whether TTM can execute the prevention and

treatment of CCl4-induced acute liver injury through the functional status of autophagy also

needs to be further explored.

In conclusion, TTM has a protective effect against acute liver injury through reducing

liver cell apoptosis rather than promoting liver cell proliferation. This study provides the

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experimental basis for TTM application in acute liver injury to extend its application fields as

a novel drug against acute liver injury.

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Acknowledgements

This study was finically supported by grants from Hubei Province Scientific Research

Foundation (D200729005), Chutian Scholar Program from Education Department of Hubei

Province, Hubei Superior Discipline Groups of Physical Education and Health Promotion and

Innovative Start-up Foundation from Wuhan Sports University to NC. The authors would like

to express their gratitude to the School of Basic Medical Sciences from Wuhan University for

providing a partial experimental platform.

Conflict of interest

All authors have declared no conflict of interest associated with this project.

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Figure legends

Figure 1. TTM rescued CCl4-induced acute liver damage based on examination of ALT (A)

and AST (B) levels. All data were expressed as Mean ± SD (n = 6). #P < 0.05 and

##P < 0.01

compared with the control group; *P < 0.05 and **P < 0.01 compared with CCl4-induced

acute liver injury model group.

Figure 2. TTM inhibited CCl4-induced hepatomegaly (A) and reduced liver index (B) as well

as alleviated CCl4-induced cell necrosis or cell death (C) through the evaluation by HE

staining.

Figure 3. TTM reduced inflammatory responses from CCl4-induced acute liver injury in rats

through the determination of mRNA expression of TNF-α (A) and IL-6 (B) by RT-PCR. All

data were expressed as Mean ± SD (n = 3) from three independent experiments. #P < 0.05 and

##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the

CCl4-model group.

Figure 4. TTM executed hepatoprotection through inhibited apoptosis. TTM could obviously

reduce liver cell apoptosis of rats induced by CCl4 based on the TUNEL staining (A) and its

statistical analysis (B). Similarly, TTM could result in down-regulation of Bax and Caspase-3

and up-regulation of Bcl-2 based on western blot analysis (C). ##P < 0.01 compared with the

control group; *P < 0.05 and

**P < 0.01 compared with the CCl4-model group.

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Figure 5. Effect of TTM on liver cell regeneration in the presence of CCl4 induction.

Compared with the CCl4-induced model group, DDB could promote liver cell regeneration

but TTM at various dosages did not exhibit the promotion role in liver cell regeneration,

which was evaluated by Ki67 staining (A) and corresponding statistical analysis (B). ##P <

0.01 compared with the control group; **P < 0.01 compared with the CCl4-induced model

group.

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Figure 1

86x64mm (300 x 300 DPI)

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Figure 2

85x63mm (300 x 300 DPI)

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Figure 3

86x64mm (300 x 300 DPI)

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Figure 4

86x64mm (300 x 300 DPI)

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Figure 5

86x64mm (300 x 300 DPI)

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